Chronic rhinosinusitis (CRS) is a confusing disease for both allergists and otorhinolaryngologists, partially due to its poorly understood pathophysiology and partially due to its limited treatment options. Several recent reports now provide evidence for a better understanding of the etiology and the relationship of CRS to airborne fungi, especially to Alternaria . First, the development of novel methods enables detection of certain fungi in mucus from the nasal and paranasal sinus cavities. Second, a non-immunoglobulin E-mediated immunologic mechanism for reactivity of CRS patients to certain common fungi has been described. Third, these fungi are surrounded by eosinophils in vivo, suggesting that they are targeted by eosinophils. Fourth, the preliminary results of studies using antifungal agents to treat patients with CRS are promising. Overall, these recent discoveries provide a logical mechanism for the pathophysiology of CRS, and they also suggest promising avenues for treatment of CRS with antifungal agents.

Introduction A current report by the Centers for Disease Control and Prevention estimates that the prevalence of sinusitis is up to 14.1% (29.2 million) of the adult US population, and the fact that there is no US Food and Drug Administration (FDA)-approved treatment for this disorder emphasizes the profound impact of this disease [1,2]. Patients with chronic rhinosinusitis (CRS) suffer from long-term nasal congestion, thick mucus production, loss of sense of smell, and intermit­tent acute exacerbations secondary to bacterial infections [1,3]. All of these symptoms can impair the patient's quality of life more severely than that of patients with congestive heart failure [4]. In recent years, it has become clear that to understand CRS, one needs to examine the inflammation that exists within and outside the hyperplastic tissue.

Understanding the Inflammation CRS is characterized histologically by an intense eosino­philic infiltration into the nasal mucosa [5,6,7,8]. This eosinophilic inflammation occurs with or without nasal polyposis, is independent from atopy, and is present neither in healthy controls nor in patients with acute bacterial or viral sinus infections. The inflammation can be easily over­looked if patients are given systemic steroids or other anti­inflammatory medication before the tissue is harvested for examination. This distinctive eosinophilic inflammation is also very heterogeneous (ie, without eosinophilic infiltra­tion in one area of a nasal mucosal tissue specimen, but with intense eosinophilic infiltration in another area of the same specimen) [9]. Thus, reports in which only single biopsies are examined and in which it was unclear whether patients had received steroids before the biopsies were taken need to be interpreted carefully regarding the intensity of the eosinophilic infiltrate. In addition, granule proteins from the eosinophils, such as major basic protein (MBP), that are toxic to sinus epithelium have been co-localized with the epithelial damage found in CRS [7,10]. In recent in vivo studies, mostly intact eosinophils were observed in the nasal tissues, but in the mucus, the eosinophils formed clusters, degranulated, and released their MBP at estimated levels far exceeding those needed to damage the epithelium [11]. These in vivo observations explain the patterns of damage in CRS, where only the outer layers of tissue are damaged, suggesting that the damage to the epithelium is inflicted from the outside (luminal side). This epithelial damage may predispose CRS patients to be susceptible for the secondary bacterial infections, leading to acute exacerbations, which are observed clinically. Because bacteria typically elicit a neutrophilic inflammation, these acute exacerbations of CRS are presumed to be of bacterial origin. However, the underlying eosinophilic inflammation that predominates in CRS is unlikely to be caused by bacterial infection, suggest­ing a nonbacterial etiologic mechanism for CRS.

Eosinophilic inflammation has been observed in tissues that contain large, nonphagocytosable parasites- eg, helminthes [12]. Earlier reports documented the accu­mulation of eosinophils and their subsequent degranula­tion on the surfaces of the parasites. The toxic proteins in the granule (including MBP) damage and kill the organ­isms. Recent observations of eosinophil clusters in mucus from CRS patients are reminiscent of the accumulations around parasites [11,13-15].

Figure 1. A, An example of an eosinophil cluster in the mucin of a chronic rhinosinusitis patient in which eosinophils appear to target fungal organisms. This specimen was attached to sinus tissue and was removed during surgery. Due to the heterogeneous nature of the eosinophilia, where areas with and without eosinophils coexist, these clusters can be easily missed on examination (hematoxylin and eosin counterstain of the same section shown in E ; magnification, ×800). B, Serial section of the same cluster shown in A , stained with GMS. The black arrow identi­fies a fungal organism (magnification, ×800). C, Serial section of the same cluster shown in A and B , stained for chitinase by immunofluores­cence. Fungal elements ( white arrows ) are evident within the eosinophil cluster (magnification, ×800). D, Serial section of the same cluster shown in A-C , stained for Alternaria by immunofluorescence. Staining occurs both within and around fungal organisms (magnification, ×800). E, Same section as that shown in A , stained for eosinophil granule major basic protein (MBP) by immunofluorescence. Extracellular deposition of the toxic eosinophil granule protein MBP is evident within the eosinophil cluster (magnification,

Two prospectively designed histologic studies of mucus obtained during CRS surgery used extra caution to preserve the mucus. Eosinophilic mucus with clusters of aggregated eosinophils was found in 96% (97/101) and 94% (35/37) of consecutive CRS patients [14,15]. Another study dem­onstrated that eosinophils released their toxic MBP in the mucus within these clusters, and not in the tissue [11]. Estimated concentrations of MBP within the clusters, based on digital analysis of the intensity of the MBP staining, were as high as 2 mM and exceeded those capable of medi­ating epithelial damage. Overall, the clusters of eosino­phils and intense eosinophil degranulation in the mucus suggest that eosinophils move from tissue to mucus with the fungi as their targets. (Fig. 1)

A key question is whether the eosinophils in CRS play a defensive role similar to the one they play against parasites in their accumulation around extramucosal fungi. Stated another way, are eosinophils recruited to target fungi in the mucus of the CRS patient? To answer this question, it is necessary to study whether certain fungi induce the recruit­ment, activation, survival, and degranulation of eosino­phils from CRS patients, but not from healthy controls.

Where Are Fungi?
The role for fungi in CRS is noninvasive, and it is not a fungal infection. In fact, it needs to be differentiated from other forms of fungal sinusitis, such as fungus balls (non­invasive) and invasive fungal sinusitis (acute fulminant or chronic form). The fungi in CRS patients are found only in the nasal mucus. Recently, striking progress has been made in the development of better techniques to detect fungi in the nasal secretions. To improve the sensitivity of fungal culture techniques, adequate amounts of mucus need to be harvested, and the disulfide bridges need to be chemically broken in the mucin to release the entrapped fungi [14,15]. Using these novel culturing techniques, newer studies demonstrated the presence of fungi in 96% ( n = 202) and 91.3% ( n = 92) of mucus from random CRS patients [14,15]. For the first time, these techniques revealed the presence of fungi in almost every healthy control; this is not surprising, given the ubiquity of fungi in the air. Finally, fungi have been found in nasal secretions of healthy infants [16]. Immediately after birth, 20% of babies showed positive fungal cultures in their nasal secretions; at 5 days, 15% showed positive cultures; at 2 months, 72% showed positive cultures; and at 4 months, 94% showed positive cultures.

Increased awareness among pathologists and surgeons coupled with new techniques for mucus collection has resulted in higher detection rates of fungi on histology. An earlier comparison study showed a 47% failure rate in the demonstration of fungi and eosinophilic mucin in patients who were suspected for "allergic fungal sinusitis" (AFS) [17]. More recently, with careful collection of nasal mucus speci­mens from CRS patients and with Gomori's methamine 474 Sinusitis

silver stain (GMS), 82% ( n = 101) and 75% ( n = 82) of CRS patients showed fungal elements in their mucus [14,15]. With the use of a chitin-based immunofluorescence staining technique, it was found that 100% of nasal mucus speci­mens contained fungal elements [18]. Overall, the newer techniques for fungal detection demonstrate increased spec­ificity and sensitivity over old techniques in detecting the presence of fungi.

Other techniques have been used by researchers to further enhance the detection of fungi. Polymerase chain reaction (PCR) with specific fungal primers has been used to find fungal DNA in polypoid nasal tissues from 100% of CRS patients ( n = 27) [19]. The authors speculated that this might represent fungal DNA that is being processed for antigen presentation. Interestingly, Alternaria -specific DNA was present in tissues from CRS patients, but not in tissues from healthy controls. Immunoassays detected levels of Alternaria antigens (ALT-a1) and total Alternaria proteins in the mucus of 100% of CRS patients as well as in the mucus from all healthy controls [20,21]. These results are not surprising in view of a new study from the National Institute of Environmental Health Science, which reports that virtually everyone is exposed to Alternaria alternata antigens at home [22].

Because of newer collection, extraction, and detection techniques, fungi, and specifically Alternaria species, are found to be present in the nasal secretions of both CRS patients and healthy controls. Thus, the mere presence of fungi in the nasal and paranasal secretions does not seem to induce a chronic eosinophilic inflammation, nor is the presence of fungi alone diagnostic of a disease. However, to understand the presence of these organisms or their products, it is necessary for one to hypothesize that the inflammation in CRS might be due to aberrant immune responses directed against certain fungi.

Why Do Eosinophils Exist? The Immune Response to Fungi Although some CRS patients produce specific immuno­globulin E (IgE) against fungi [23,24], there is no evidence that this IgE production directly results in the disease state of CRS. Furthermore, CRS develops in patients who have IgE antibodies to fungi or other common aeroallergens and in those who do not. In contrast, IgE-mediated allergy and exposure to the relevant allergens leads to allergic rhinitis. Thus, patients with CRS may have IgE-mediated hypersensi­tivity to molds as a comorbid disease, but the underlying eosinophilic inflammation appears to be driven by a mecha­nism independent from an IgE-mediated one. If the human immune system in CRS patients recognizes these fungi as foreign and uses eosinophils to attack them, one could speculate that it should recruit and activate eosinophils by production of cytokines that regulate eosinophil inflamma­tion. Tissue-bound lymphocytes have been found to be the main source of these cytokines in patients with CRS [25].

In addition, the expression of vascular cell adhesion molecule-1 (VCAM-1) has been identified in the vascular endothelium in CRS patients [26]. This expression occurred independent of any IgE-mediated allergy and explains the presence of eosinophils in allergic as well as nonallergic patients with CRS [26]. VCAM-1 is known to specifically bind to the VLA-4 (very late-appearing antigen-4) on eosino­phils, thus causing selective adhesion and migration of eosinophils from the vasculature to the sinus tissue [26]. Shin et al. [20] recently demonstrated that isolated periph­eral blood mononuclear cells (PBMCs) from CRS patients, which contained lymphocytes and other cells that can serve as antigen-presenting cells, produced large amounts of interleukin (IL)-13 when exposed in vitro to certain mold extracts, especially from Alternaria spp. This produc­tion of IL-13 in response to Alternaria may enhance expres­sion of VCAM-1 by vascular endothelial cells [27].

Significantly elevated levels of IL-5, a cytokine that mediates eosinophil differentiation, survival, and activa­tion, are present in tissue specimens of CRS patients and not in those of healthy controls [28-33]. A majority of the IL-5 staining cells are lymphocytes (68%), followed by eosinophils (18%) and mast cells (14%) [34]. PBMCs from 16 out of 18 CRS patients stimulated with Alternaria antigens in vitro show increased production of IL-5, but PBMCs from 15 out of 15 healthy controls do not [20]. Elevated levels of specific IgE for Alternaria were detected in only 28% of these CRS patients; the increased IgE levels did not correlate with increased levels of IL-5 [20]. PBMCs from allergic and nonallergic CRS patients produced similar amounts of IL-5, indicating that this reaction is independent from an IgE-mediated allergic reaction [20]. In addition, PBMCs from CRS patients stimulated with either Cladosporium (6/18) or Aspergillus (4/18) antigens also show increased production of IL-5; no response is seen with Penicillium antigen [20]. Furthermore, production of interferon-? (IFN-?), which facilitates destruction of parasites by eosinophils, is 5.5 times higher in PBMCs from CRS patients stimulated with Alternaria antigen compared with production by healthy control PBMCs [20]. These differences in cytokine pro­duction probably cannot be explained by differences in fungal contents in nasal mucus. When nasal secretions from nine healthy controls and nine CRS patients were examined, there were no differences in their levels of total Alternaria proteins [20].

However, there are differences between CRS patients and healthy controls in their immune responses to fungi. Mean serum IgG levels specific for Alternaria were increased about fivefold in the 18 CRS patients compared to the 15 healthy controls [20]. Furthermore, serum IgG levels in the CRS patients directly correlated with the levels of IL-5 produced when patients' PBMCs were incubated with Alternaria [20]. Given the general notion that the levels of IgG indicate the amount of immunologic exposure, these results suggest a direct correlation between

the exposure to Alternaria antigens and the severity of the immune reaction as determined by the amount of IL-5 production. Other investigators also found an increase of nasal obstruction after challenge with Alternaria that was independent from an IgE-mediated hypersensitivity [35]. In a recent study, eosinophils from healthy people that were incubated with Alternaria and Penicillium antigens released significant amounts of eosinophil-derived neuro­toxin, a marker of eosinophil degranulation [36]. Other fungal antigens, including Aspergillus , Cladosporium , and Candida , did not induce eosinophil degranulation, suggest­ing the presence of an innate immune response to certain fungi in human. Thus, both innate and acquired immune responses to environmental fungi, such as Alternaria (inde­pendent of IgE antibodies to Alternaria ) may increase production of the cytokines and provide cellular activation signals necessary for the robust eosinophilic inflammation in CRS patients.

Antifungal Treatment of Chronic Rhinosinusitis Given the recent understanding of the association of numerous eosinophils with fungi in the mucus of CRS patients, it now appears that antifungal agents applied directly to the nasal mucus could be beneficial. By reducing the fungal burden, the eosinophilia and accompanying inflammation might also be attenuated.

In one open-label pilot study, topical intranasal treat­ment of 51 CRS patients, given as 20 mL of a 100 µg/mL amphotericin B solution per nostril, twice a day for an average of 11.3 months (range 3-17 months), improved both symptoms and endoscopic staging in 75% of patients and reduced mucosal thickening on available CT scans [37]. Ricchetti et al. [38] reported disappearance of polypo­sis on endoscopy in 62% of mild and 42% of moderate patients with CRS when topical amphotericin B was used for 4 weeks. However, patients with severe CRS, who had polyps that filled the entire nasal cavity, showed no improvement [38]. In this severe CRS group, failure might be due to the limited access of the topical medication or to the short duration of the therapy in the study, or both [39]. Likewise, a trial with CRS patients, who also had severe nasal polyposis, used small volumes of amphotericin B, and found no benefit after 8 weeks [40]. When a bulb syringe was used as a delivery vehicle, a double-blinded, placebo-controlled trial of intranasal amphotericin B in an unselected CRS population found efficacy compared to placebo after 6 months [21]. Reduced inflammatory mucosal thickening on CT scan and nasal endoscopy and decreased levels of intranasal cytokines and markers for eosinophilic inflammation in CRS patients could be dem­onstrated. Recently, a group of CRS patients was success­fully treated with steroids and itraconazole in an open-label study, and no surgery was needed [41].

These results suggest that an antifungal treatment may reduce the fungal antigenic load in the nasal and paranasal cavities and subsequently decrease the eosinophilic response. Overall, topical antifungal treatments likely benefit patients with CRS; this treatment needs to be long-term; and the dos­age, formulation, and application methods still need further optimization, specifically in those with severely obstructive nasal and sinus passages.

A Broader Role for Fungi in Chronic Rhinosinusitis New data have been presented that highlight the importance of certain airborne fungi, in particular the Alternaria species, in the pathophysiology of CRS. Although newer detection techniques have demonstrated the presence of these fungi in virtually everyone, only patients with CRS may react to them with the production of cytokines, which are crucial for the eosinophilic inflammation. In addition, Alternaria extract induces a striking degranulation of eosinophils in vitro. Eosinophils apparently target fungi in vivo in the mucus of CRS patients, where they degranulate within clusters. This extramucosal release of cytotoxic proteins may explain the damage observed to the epithelium, which is inflicted from the outside (luminal side), and the susceptibility of CRS to secondary bacterial infection (acute exacerbations of CRS). Antifungal therapy, if used in the correct formulation, appli­cation form, and duration, may be effective by inhibiting the antigenic load with a subsequent reduction of intranasal cytokines and markers of eosinophilic inflammation.

This study showed that in CRS patients, eosinophils form clusters in the mucus where they release MBP, which is diffusely deposited on the epi­thelium, a process not observed in the tissue. Estimated MBP levels far exceed those needed to damage epithelium. The damage was from the luminal side and could explain the secondary bacterial infections.